1. Field of the Invention
This invention relates to inhibiting the production of cytokines in cells of the body, and thus to the treatment or prevention of diseases associated with excessive production of cytokines. Of especial interest is the production of interleukin-5, a cytokine which is implicated in inflammation of the pulmonary airways, especially in asthma; and TNF-xcex1, a cytokine shown to play a very important role in rheumatoid arthritis.
2. Description of the Related Art
There is now evidence that interleukin-5 (IL-5) plays an important role in the pathogenesis of asthma (Corrigan, Clin. Exp. Allergy 25, 485-487 (1995)). IL-5 is one of the commonest cytokines which are produced by T-helper cells. It is particularly implicated in control of eosinophil functions, including selective chemotaxis, growth differentiation, proliferation, release of cytotoxic proteins and the survival of these cells (Takatsu et al., Adv. in Immunol. 57, 145-190 (1995)). IL-5 also enhances T-cell adhesion to vascular endothelial cells. IL-5 has been detected at the site of the allergic inflammation and in the peripheral blood of patients, its level correlating to the extent of eosinophilia, the severity, and the response of the treatment of asthma (H. Okudaira et al., Int. Arch. Allergy Immunol. 107, 255-258 (1995); Corrigan, Clin. Exp. Allergy 25, 485-487 (1995)). Animal models from murine to monkey have demonstrated the involvement of IL-5 in asthma and the beneficial effects due to the inhibition of the cytokine.
A prophylactic and therapeutic effect of an anti-IL-5 monoclonal, TRFK-5, in a mouse model of allergic pulmonary inflammation has recently been demonstrated (T. T. Kung et al., American Journal of Respiratory Cell and Molecular Biology 13, 360-365 (1995)). The number of eosinophils in the bronchoaveolar lavage (BAL) fluid and lung tissue was reduced and the decrease in bone marrow eosinophils was prevented. Both these effects were being seen to occur in a dose-dependent fashion. After the neutralisation of IL-5, the ovalbumin-allergic mice showed no evidence of increased epithelial damage, oedema, or the presence of mucus that could have resulted from eosinophil apoptosis and release of toxic proteins. In IL-5xe2x80x9cknock-outxe2x80x9d mice, the airway pathology was abolished (P. S. Foster et al., J. Exp. Med. 183, 195-201 (1996)).
Other groups have worked on guinea pig models and have reported that in studies using anti-IL-5 antibodies, a total inhibition of the development of bronchohyperreactivity to histamine and arecoline after ovalbumin challenge (A. J. M. Van Oosterhout et al., American Review of Respiratory Disease 147, 548-552 (1993)). The number of eosinophils was markedly reduced and the number of neutrophils was not affected, verifying the specificity of IL-5 on eosinophils in asthma. However, antibody therapy in humans has many well-known problems.
An alternative idea which could be contemplated is to inhibit production of IL-5 prior to RNA transcription using a modified synthesised double stranded (msds) DNA sequence to which is similar or identical to a particular part of the promoter region of IL-5, for example the part which binds to a transcription factor.
As a general principle, a DNA xe2x80x9cdecoyxe2x80x9d would be provided which would compete with the native gene for a limited supply of transcription factor. This would be a double-stranded (ds) DNA oligomer modified to prevent it from being destroyed by endogenous DNases. For example, the phosphate linkages could be replaced to a large extent by phosphorothioate linkages. See Bieliaska et al., Science 250, 997-1000 (1990), in relation to interleukin-2.
NOMENCLATURE NOTE
All 5xe2x80x2-non-coding region nucleotide nomenclature used in this patent specification is based on the first nucleotide of the start of the coding region (A of the ATG) being +1 and the nucleotide preceding it xe2x88x921. All nomenclature used in references has been converted to this basis.
V. Gruart-Gouilleux et al., Eur. J. Immunol 25, 1431-1435 (1995) studied the transcriptional activity of the human IL-5 gene promoter region in mouse EL4 cells, using a series of deletion constructs linked to a luciferase reporter gene. They found that deletions of nucleotides between xe2x88x92358 and xe2x88x92274 or xe2x88x92124 to xe2x88x9279 inhibited the induction of IL-5 promoter activity. Investigation of the xe2x88x92358 to xe2x88x92274 region revealed that the octamer-binding motif 5xe2x80x2-atgcaaat-3xe2x80x2 at xe2x88x92290 to xe2x88x92283 was required for full transcriptional activity. See nomenclature note above. Very minor errors of numbering in this paper have also been corrected, taking the sequence of oligo probes as correct.
At about the same time D. Z. Staynov et al., Proc. Natl. Acad. Sci. USA 92, 3606-3610 (1995) and Int. Arch. Allergy Immunol. 107, 217-219 (1995) described a regulatory element in the promoter of the human granulocyte-macrophage colony-stimulating factor (hGMCSF) gene that has related sequences in the gene promoters of human interleukins (hIL-2, hIL-4, hIL-5, hIL-13). The authors identified a palindrome-containing sequence as involved in a DNA-protein interaction, by using nuclear extract from Jurkat cells as the source of protein. A 40 bp sequence of hGMCSF promoter DNA containing cttgg . . . (22N) . . . ccaag xe2x80x9couterxe2x80x9d palindromic sequences near each end and a 12 bp-long xe2x80x9cinnerxe2x80x9d, AT-rich, palindrome in the centre was found to bind to the nuclear extract by a gel retardation assay (in which the protein-DNA complex migrates more slowly than the DNA alone on a gel). Some of the binding was ascribed exclusively to the outer palindrome and some to the inner. However, the use of these crude nuclear extracts calls into question which proteins were bound. Noting that the outer palindrome motif or a shorter version, ttgg . . . ccaa, is present in the promoters of certain interleukins, the authors speculated that some of the interacting proteins may be gene-specific. As regards IL-5, the conterpart palindrome-containing sequence occurs at nucleotides xe2x88x92514 to xe2x88x92504. However, V. Gruart-Gouilleux et al., above, reported that a deletion of nucleotides xe2x88x92552 to xe2x88x92449 (thus encompassing the whole of Staynov et al.""s palindrome-containing sequence) had little effect on induction of IL-5 promoter activity.
It has now surprisingly been found that a double-stranded oligomeric deoxyribonucleotide (oligo-DNA), modified to make it resistant to degradation, containing a similar palindrome in hIL-5, inhibits production of hIL-5 in T-cells, apparently by competing with hIL-5 for a transcription factor. It has further been found that there are palindrome-containing sequences comprising the ttgg . . . ccaa motif in the gene promoter region of other cytokines besides those mentioned by Staynov et al., such as TNF-xcex1. These are also inhibitors of transcription of the specific cytokines. The inhibition referred to herein comprises partial or complete prevention of the transcription.
By delivering these oligonucleotides to cells, it will be possible to treat diseases characterised by over-expression of a target gene, leading to unwanted effects at the cellular level, at the level of tissue or organ function and/or throughout the body. These include but are not limited to allergy, asthma, chronic obstructive airways disease or conditions characterised as being inflammation, including autoimmune diseases, or cancer.
In one aspect, the present invention provides a ds-DNA oligomer which has from 25 to 150 base pairs, preferably 25 to 60 and more preferably 50 base pairs, and comprises (i.e. consists of or includes) the palindrome-containing sequence (one strand only shown) of formula 1:
5xe2x80x2B1. . . xN . . . B23xe2x80x2xe2x80x83xe2x80x83(formula 1)
wherein B1 is ttgg or ccaa and B2 is correspondingly ccaa or ttgg and xN represents a sequence of from 1 to 30 intervening nucleotides, preferably 3 to 30 nucleotides, which is substantially native to the promoter region of the gene coding for a cytokine, said intervening nucleotides being the same or different and optionally comprising (i.e. consisting of or including) palindromic sequence, together with the complementary strand, the oligomer being modified to make it resistant to degradation and denaturing in vivo. Preferably the oligomer is terminated by stability-imparting c or g nucleotides at each end. It can be made degradation-resistant in any of the known ways, especially by replacing 50-100% of phosphate bonds therein by phosphorothiorates. An alternative method is to clone the oligomer(s) into a plasmid.
The invention is not necessarily applicable to every single cytokine, but only to those which have the required sequence of formula (1). As will be seen later, this includes most of the best known human cytokines, especially those which are produced by T-helper (Th) cells. However, it also applies to certain other cytokines, such as those produced by macrophages.
Generally stated, the cytokines to which the present invention applies can easily be determined by searching for a palindrome-containing sequence of formula (1). Within the promoter region, or if the promoter region is not yet identified, it should be regarded as the first 3000 bases, especially the first 1000 bases, of 5xe2x80x2-non-coding region preceding the coding region, i.e. upstream of ATG. By definition, the promoter region is also upstream of the start site for transcription of RNA, and therefore that part of the 5xe2x80x2-non-coding region which is transcribed to mRNA or is an intron of the gene is excluded. Extensive lengths of promoter sequence have been published for many cytokines, so sequencing will rarely be necessary.
As mentioned earlier, the invention also includes a vector containing at least one copy of a ds-DNA palindrome sequence (one strand only shown) of formula (1) above, together with the complementary strand. The vector may be for example a plasmid or a phage or a modified viral vector. This approach has the advantage of permitting multiple copies of the DNA sequence, e.g. up to 100, to be administered and provides more stability in the form of a circular plasmid/vector.
The invention also includes pharmaceutical formulations of the above-defined DNA or vector, especially liposomes containing the DNA or vector.
Also within the invention is the medical use of the above DNA, expressed in any way permitted by patent law. Thus, in the United States and Australia it includes a method of treating a disease associated with over-production of a cytokine especially by over-expression of the gene, the method comprising the administration to a patient in need thereof of an effective amount of the DNA oligomer, vector or pharmaceutical formulation as defined above. For Europe and those countries which adopt European Patent Convention or similar law, it includes the DNA, vector and pharmaceutical formulation, for use in the therapy of the said disease and the use of the DNA or vector in the preparation of a pharmaceutical formulation for that purpose.